Process of preparing a peptide epoxyketone immunoproteasome inhibitor, and precursors thereof

ABSTRACT

Disclosed herein are methods for preparing [(2S,3R)-N-[(2S)-3-(cyclopent-1-en-1-yl)-1-[(2R)-2-methyloxiran-2-yl]-1-oxopropan-2-yl]-3-hydroxy-3-(4-methoxyphenyl)-2-[(2S)-2-[2-(morpholin-4-yl)acetamido]propanamido]propanamide (compound “G”): 
     
       
         
         
             
             
         
       
     
      and precursors thereof.

TECHNICAL FIELD

The disclosure relates to methods and processes of preparing(2S,3R)-N-[(2S)-3-(cyclopent-1-en-1-yl)-1-[(2R)-2-methyloxiran-2-yl]-1-oxopropan-2-yl]-3-hydroxy-3-(4-methoxyphenyl)-2-[(2S)-2-[2-(morpholin-4-yl)acetamido]propanamido]propanamide,and precursors thereof.

DESCRIPTION OF RELATED TECHNOLOGY

The compound,(2S,3R)-N-[(2S)-3-(cyclopent-1-en-1-yl)-1-[(2R)-2-methyloxiran-2-yl]-1-oxopropan-2-yl]-3-hydroxy-3-(4-methoxyphenyl)-2-[(2S)-2-[2-(morpholin-4-yl)acetamido]propanamido]propanamide(“compound G”), is useful as an immunoproteasome inhibitor:

In eukaryotes, protein degradation is predominately mediated through theubiquitin pathway in which proteins targeted for destruction are ligatedto the 76 amino acid polypeptide ubiquitin. Once targeted, ubiquitinatedproteins then serve as substrates for the 26S proteasome, amulticatalytic protease, which cleaves proteins into short peptidesthrough the action of its three major proteolytic activities. Whilehaving a general function in intracellular protein turnover,proteasome-mediated degradation also plays a key role in many processessuch as major histocompatibility complex (MHC) class I antigenpresentation, apoptosis, cell growth regulation, NF-κB activation,antigen processing, and transduction of pro-inflammatory signals.

The 20S proteasome is a 700 kDa cylindrical-shaped multicatalyticprotease complex comprised of 28 subunits organized into four rings. Inyeast and other eukaryotes, 7 different a subunits form the outer ringsand 7 different β subunits comprise the inner rings. The α subunitsserve as binding sites for the 19S (PA700) and 1 IS (PA28) regulatorycomplexes, as well as a physical barrier for the inner proteolyticchamber formed by the two β subunit rings. Thus, in vivo, the proteasomeis believed to exist as a 26S particle (“the 26S proteasome”). In vivoexperiments have shown that inhibition of the 20S form of the proteasomecan be readily correlated to inhibition of 26S proteasome. Cleavage ofamino-terminal prosequences of β subunits during particle formationexpose amino-terminal threonine residues, which serve as the catalyticnucleophiles. The subunits responsible for catalytic activity inproteasomes thus possess an amino terminal nucleophilic residue, andthese subunits belong to the family of N-terminal nucleophile (Ntn)hydrolases (where the nucleophilic N-terminal residue is, for example,Cys, Ser, Thr, and other nucleophilic moieties). This family includes,for example, penicillin G acylase (PGA), penicillin V acylase (PVA),glutamine PRPP amidotransferase (GAT), and bacterialglycosylasparaginase. In addition to the ubiquitously expressed βsubunits, higher vertebrates also possess three interferon-y-inducible βsubunits (LMP7, LMP2 and MECL1), which replace their normalcounterparts, B5, Bl and B7 respectively, thus altering the catalyticactivities of the proteasome. Through the use of different peptidesubstrates, three major proteolytic activities have been defined for theeukaryote 20S proteasome: chymotrypsin-like activity (CT-L), whichcleaves after large hydrophobic residues; trypsin-like activity (T-L),which cleaves after basic residues; and peptidylglutamyl peptidehydrolyzing activity (PGPH), which cleaves after acidic residues. Twoadditional less characterized activities have also been ascribed to theproteasome: BrAAP activity, which cleaves after branched- chain aminoacids; and SNAAP activity, which cleaves after small neutral aminoacids. The major proteasome proteolytic activities appear to becontributed by different catalytic sites, since inhibitors, pointmutations in β subunits and the exchange of γ interferon-inducing βsubunits alter these activities to various degrees.

PCT publication no. WO 2014/152134, which is incorporated herein byreference, describes tripeptide epoxy immunoproteasome inhibitors, suchas Compound G, and methods for their small-scale synthesis. However, alarge-scale synthesis of tripeptide epoxy immunoproteasome inhibitors,such as Compound G, is needed for commercial development.

SUMMARY OF THE INVENTION

In one aspect, the disclosure provides a method of preparing(2S,3R)-N-[(2S)-3-(cyclopent-1-en-1-yl)-1-[(2R)-2-methyloxiran-2-yl]-1-oxopropan-2-yl]-3-hydroxy-3-(4-methoxyphenyl)-2-[(2S)-2-[2-(morpholin-4-yl)acetamido]propanamido]propanamide(compound “G”)

comprising:

-   (a) admixing a tertiary amine base and a suspension of:    -   (i)        (2S,3R)-3-hydroxy-3-(4-methoxyphenyl)-2-((S)-2-(2-morpholino-acetamido)propanamido)propanoic        acid (compound “E”):

    -   

    -   and

    -   (ii) (S)-3-(cyclopent-1-en-1-yl        )-1-((R)-2-methyloxiran-2-yl)-1-oxopropan-2-aminium salt        (compound “F”):

    -   

    -   wherein X⁻ is a counterion; in an aprotic solvent to form a        mixture; and-   (b) admixing a coupling agent and the mixture of step (a) to form    compound G; wherein the temperature of each admixing step is    maintained at -20° C. to 25° C.

In some embodiments, X⁻ is selected from the group consisting oftosylate, triflate, acetate, naphthalene sulfonate,4-nitrobenzenesulfonate, sulfate, methylsulfate, nitrate, fluoride,chloride, bromide, and combinations thereof. In some cases, wherein X⁻is tosylate, naphthalene sulfonate, or 4-nitrobenzenesulfonate. Forexample, X⁻ is tosylate.

In various embodiments, the aprotic solvent is selected from the groupconsisting of acetonitrile (“ACN”), dichloromethane (“DCM”),tetrahydrofuran (“THF”), dimethylacetamide (“DMAc”), ethyl acetate(“EtOAc”), isopropyl acetate (“iPrOAc”), dimethylformamide (“DMF”), andcombinations thereof. For example, the aprotic solvent can be DCM.

In some cases, the tertiary amine base is selected from the groupconsisting of N,N-diisopropylethylamine (“DIPEA”), triethylamine(“TEA”), N-methylmorpholine (“NMM”), 2,2,6,6-tetramethylpiperidine(“TMP”), 2,4,6-trimethylpyridine (“collidine”), and combinationsthereof. For example, the tertiary amine base can includes DIPEA. Invarious cases, the molar ratio of the tertiary amine base to compound Eis in a range of 1:1 to 4:1.

In some embodiments, the coupling agent comprises a carbodiimidereagent, a phosphonium reagent, a uronium reagent, an immonium reagent,an imidazolium reagent, an organophosphorus reagent, an acid chloridereagent, a chloroformate reagent, or a pyridinium reagent. In variousembodiments, the uronium reagent is selected from the group1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid hexafluorophosphate (“HATU”), O-(Benzotriazol-l-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate (“HBTU”), and combinationsthereof. For example, the uronium reagent can be HATU. In some cases,the molar ratio of coupling agent to compound E is 1 to 1. The couplingreagent further comprises a coupling additive. In some embodiments, thecoupling additive is selected from the group consisting of abenzotriazole, a dicarboximide, a succinimide, and combinations thereof.In various embodiments, the coupling additive is selected from the groupconsisting of N-hydroxysuccinimide (“HOSu”),N-hydroxy-5-norbomene-2,3-dicarboximide (“HONB”), 1-hydroxybenzotriazole(“HOBt”), 6-chloro-1-hydroxybenzotriazole (“Cl-HOBt”),1-hydroxy-7-azabenzotriazole (“HOAt”), and combinations thereof.

In various cases, the temperature of each admixing step is maintained at-15° C. to 25° C. In some cases, the admixing of step (a) comprisesstirring the mixture for up to 10 minutes. In various embodiments, theadmixing of step (b) comprises stirring for up to two hours. In someembodiments, compound G is washed with one or more of the following:water, potassium phosphate monobasic, sodium bicarbonate, and sodiumsulfate.

In various embodiments, compound E is prepared by admixing a reductantand benzyl(25,3R)-3-hydroxy-3-(4-methoxyphenyl)-2-((5)-2-(2-morpholinoacetamido)propanamido)propanoate(compound “D”)

to form compound E. In some cases, the reductant is selected from thegroup consisting of H₂, Pd/C; H₂, Pd(OH)₂/C; Li; Na; lithium4,4’-di-tert-butylbiphenyl (“Li DTBBP”), and combinations thereof. Insome embodiments, the admixing of the reductant and compound D occursunder a nitrogen atmosphere. The admixing of the reductant and compoundD can occur for up to 4 hours. Further, the admixing can occur at atemperature in a range of 10° C. to 20° C. In various cases, thepreparation of compound E further includes one or more of the following:filtering compound E across diatomite; washing compound E; andcrystallizing compound E with THF and water.

Another aspect of the disclosure provides a method of preparing(S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methyloxiran-2-yl)-1-oxopropan-2-aminiumsalt (compound “F”)

comprising:

-   (a) admixing trifluoroacetic acid (“TFA”) and    tert-butyl-((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methyloxiran-2-yl)-1-oxopropan-2-yl)carbamate    (compound “H”):

-   

-   in an aprotic solvent at a temperature in a range of -5° C. to 5° C.    to form a mixture;

-   (b) concentrating the mixture; and

-   (c) admixing an acid and the concentrated mixture of step (b) at a    temperature in a range of -5° C. to 5° C. to form compound F,

-   wherein X⁻ is a conjugate base of the acid.

In some embodiments, the acid is selected from the group consistingofp-toluenesulfonic acid, trifluoromethanesulfonic acid, acetic acid,trifluoroacetic acid, naphthalene sulfonic acid, 4-nitrobenzenesulfonicacid, sulfonic acid, methylsulfonic acid, benzenesulfonic acid, nitricacid, HF, HCl, HBr, and combinations thereof. For example, the acid canbe selected from the group consisting of toluenesulfonic acid,naphthalene sulfonic acid, 4-nitrobenzenesulfonic acid, and combinationsthereof. In some cases, the molar ratio of the acid to compound H is 1to 1. In various cases, the molar ratio of TFA to compound H is 8 to 1.In various embodiments, the aprotic solvent in step (a) is selected fromthe group consisting of acetonitrile (“ACN”), dichloromethane (“DCM”),tetrahydrofuran (“THF”), dimethylacetamide (“DMAc”), methyl tert-butylether (“MTBE”), isopropyl ether (“IPE”), and combinations thereof. Forexample, the aprotic solvent can include DCM. In some cases, thetemperature in step (a), step (c), or both is 0° C. In various cases,the mixture of step (b) is concentrated at a temperature in a range of15° C. to 25° C. In various embodiments, the admixing of step (a)comprises stirring for 2 hours. In some cases, the admixing of step (c)comprises stirring for 10 to 12 hours. In some embodiments, theconcentrated mixture of step (b) is further washed with a polar, aproticsolvent at a temperature in a range of 15° C. to 25° C. Suitable polar,aprotic solvents include diethyl ether, tetrahydrofuran (“THF”),acetonitrile (“ACN”), methyl tert-butyl ether (“MBTE”), isopropyl ether(“IPE”) and combinations thereof. For example, the polar, aproticsolvent can include MBTE. In some cases, the method further includes oneor more of the following steps: filtering compound F, washing compound Fwith a polar, aprotic solvent, and drying compound F. The polar, aproticsolvent for washing compound F can be selected from the group consistingof diethyl ether, tetrahydrofuran (“THF”), acetonitrile (“ACN”), methyltert-butyl ether (“MBTE”), isopropyl ether (“IPE”), and combinationsthereof.

Yet another aspect of the disclosure provides a method of preparingbenzyl(2S,3R)-3-hydroxy-3-(4-methoxyphenyl)-2-((S)-2-(2-morpholinoacetamido)propanamido)propanoate(compound “D”)

comprising:

-   (a) admixing a tertiary amine base and a suspension of:    -   (i)        (2S,3R)-1-(benzyloxy)-3-hydroxy-3-(4-methoxyphenyl)-1-oxopropan-2-aminium        salt (compound “B”):

    -   

    -   wherein X⁻ is a counterion; and

    -   (ii) (2-morpholinoacetyl)-L-alanine (compound “C”):

    -   

    -   in an aprotic solvent to form a mixture; and-   (b) admixing a coupling agent and the mixture of step (a) to form    compound D;-   wherein the temperature of each admixing step is maintained at    -5° C. to 5° C.

In some embodiments, X⁻ is selected from the group consisting oftosylate, triflate, acetate, naphthalene sulfonate,4-nitrobenzenesulfonate, sulfate, methylsulfate, nitrate, fluoride,chloride, bromide, and combinations thereof. For example, X⁻ can bechloride. In some cases, the aprotic solvent is selected from the groupconsisting of acetonitrile (“ACN”), dichloromethane (“DCM”),tetrahydrofuran (“THF”), dimethylacetamide (“DMAc”), and combinationsthereof. For example, the aprotic solvent can include ACN. In variousembodiments, the tertiary amine base is selected from the groupconsisting of N,N-diisopropylethylamine (“DIPEA”), triethylamine(“TEA”), N-methylmorpholine (“NMM”), 2,2,6,6-tetramethylpiperidine(“TMP”), 2,4,6-trimethylpyridine (“collidine”), and combinationsthereof. For example, the tertiary amine base can include DIPEA. Invarious cases, the coupling agent comprises a carbodiimide reagent, aphosphonium reagent, a uronium reagent, an immonium reagent, animidazolium reagent, an organophosphorus reagent, an acid chloridereagent, a chloroformate reagent, or a pyridinium reagent. In someembodiments, the uronium reagent is selected from the group1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid hexafluorophosphate (“HATU”),O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate(“HBTU”), and combinations thereof. For example, the uronium reagent caninclude HATU. In some embodiments, the molar ratio of coupling agent tocompound B is 1 to 1. In various embodiments, the coupling reagentfurther includes a coupling additive. The coupling additive can beselected from the group consisting of a benzotriazole, a dicarboximide,a succinimide, and combinations thereof. For example, the couplingreagent can be selected from the group consisting ofN-hydroxysuccinimide (“HOSu”), N-hydroxy-5-norbornene-2,3-dicarboximide(“HONB”), 1-hydroxybenzotriazole (“HOBt”),6-chloro-1-hydroxybenzotriazole (“Cl-HOBt”),1-hydroxy-7-azabenzotriazole (“HOAt”), and combinations thereof. In somecases, the temperature of each admixing step is maintained at -5° C. to5° C. In some embodiments, the admixing of step (b) comprises mixingportions of the coupling agent with the mixture from step (a) over 30minutes. In various cases, the admixing of step (b) comprises stirring 2hours. In some embodiments, the method further includes washing compoundD with one or more of the following: water, isopropyl acetate, potassiumphosphate monobasic, sodium bicarbonate, sodium sulfate, and THF.

Compound B can be prepared by admixing (i) an acid and (ii) benzyl(2S,3R)-2-((tert-butoxycarbonyl)amino)-3-hydroxy-3-(4-methoxyphenyl)propanoate(compound “A”):

in a polar, aprotic solvent, to form compound B. In some embodiments,the acid is selected from the group consisting ofp-toluenesulfonic acid,trifluoromethanesulfonic acid, acetic acid, trifluoroacetic acid,naphthalene sulfonic acid, 4-nitrobenzenesulfonic acid, sulfonic acid,methylsulfonic acid, nitric acid, HF, HCl, HBr, and combinationsthereof. For example, the acid can include trifluoroacetic acid or HClIn some embodiments, the polar, aprotic solvent is selected from thegroup consisting of ethyl acetate, N-methylpyrrolidone (“NMP”),tetrahydrofuran (“THF”), acetone, dimethylformamide (“DMF”),acetonitrile (“ACN”), dimethyl sulfoxide (“DMSO”), dicholormethane(“DCM”), and combinations thereof. For example, the polar, aproticsolvent can include ethyl acetate, DCM, or combinations thereof. In somecases, the admixing step includes stirring at a temperature in a rangeof 15° C. to 25° C. The method can further include filtering compound B,drying compound B, or both.

Another aspect of the disclosure provides a crystalline form of(2S,3R)-1-(benzyloxy)-3-hydroxy-3-(4-methoxyphenyl)-1-oxopropan-2-aminiumchloride salt (compound “B-Cl”)

characterized by an X-ray powder diffraction pattern comprising peaks at4.6, 9.2, 13.8, 18.5, and 32.9 ± 0.2° 2θ using Cu Kα radiation.

Yet another aspect of the disclosure provides a crystalline form of(2S,3R)-3-hydroxy-3-(4-methoxyphenyl)-2-((S)-2-(2-morpholinoacetamido)propanamido)propanoicacid (compound “E”):

characterized by an X-ray powder diffraction pattern comprising peaks at6.2, 8.5, 9.7, 12.7, 13.7, 16.0, 16.9 17.2, 18.4, 18.9, 19.2, 19.7,22.5, 24.7, 25.4, 28.7, and 29.7 ± 0.2° 2θ using Cu Kα radiation.

Still another aspect of the disclosure provides a crystalline form of(S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methyloxiran-2-yl)-1-oxopropan-2-aminiumsalt (compound “F”):

wherein X⁻ is tosylate, characterized by an X-ray powder diffractionpattern comprising peaks at 6.8, 7.1, 7.4, 14.2, 14.8, 17.0, 17.5, 17.8,18.5, 18.7, 20.1, 20.3, 23.0, 23.6, 24.5, 29.3, and 31.2 ± 0.2° 2θ usingCu Kα radiation.

Further aspects and advantages will be apparent to those of ordinaryskill in the art from a review of the following detailed description.While the methods disclosed herein are susceptible of embodiments invarious forms, the description hereafter includes specific embodimentswith the understanding that the disclosure is illustrative, and is notintended to limit the invention to the specific embodiments describedherein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the characteristic differential scanning calorimetry(“DSC”) curve for(2S,3R)-1-(benzyloxy)-3-hydroxy-3-(4-methoxyphenyl)-1-oxopropan-2-aminiumsalt (compound “B”).

FIG. 2 depicts the characteristic DSC thermogram for(2S,3R)-3-hydroxy-3-(4-methoxyphenyl)-2-((S)-2-(2-morpholino-acetamido)propanamido)propanoicacid (compound “E”).

FIG. 3 depicts the characteristic thermogravimetric analysis (“TGA”)data for compound E.

FIG. 4 depicts the characteristic X-ray powder diffraction pattern(“XRPD”) for compound E.

FIG. 5 depicts the characteristic DSC thermogram for the tosylate saltof(5)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methyloxiran-2-yl)-1-oxopropan-2-aminiumsalt (compound “F”).

FIG. 6 depicts the characteristic thermogravimetric analysis (“TGA”)data for the tosylate salt of compound F.

FIG. 7 depicts the characteristic DSC thermogram for the naphthalenesulfonic acid salt of compound F.

FIG. 8 depicts the characteristic thermogravimetric analysis (“TGA”)data for the naphthalene sulfonic acid salt of compound F.

FIG. 9 depicts the characteristic XRPD pattern for the tosylate salt ofcompound F.

FIG. 10 depicts a single crystal X-ray diffraction (“XRD”) of thetosylate salt of compound F.

DETAILED DESCRIPTION

Disclosed herein is a process for the preparation of(2S,3R)-N-[(2S)-3-(cyclopent-1-en-1-yl)-1-[(2R)-2-methyloxiran-2-yl]-1-oxopropan-2-yl]-3-hydroxy-3-(4-methoxyphenyl)-2-[(2S)-2-[2-(morpholin-4-yl)acetamido]propanamido]propanamide(compound “G”):

and precursors thereof, and in some cases, the process is forlarge-scale preparation of compound G. The overall scheme for thepreparation of compound G is shown in Scheme 1, below.

The optical purity of compound G is controlled during the synthesis bythe quality of the starting materials and the specific reagents used forthe transformations.

The compounds disclosed herein may be identified either by theirchemical structure and/or chemical name herein. When the chemicalstructure and chemical name conflict, the chemical structure isdeterminative of the identity of the compound.

Unless otherwise indicated, terms and abbreviations used in thisspecification include the normal and customary meaning to those in therelevant field.

As the present disclosure’s contribution is not limited to particularembodiments or aspects disclosed herein, the disclosure provides to oneof ordinary skill in the art additional embodiments including changesand modifications to adapt to various usages and conditions. Forexample, changes and modifications to materials, methods of synthesis,or procedures described herein will be apparent to one of ordinaryskill.

When ranges are used herein for physical properties, such as molecularweight, or chemical properties, such as chemical formulae, allcombinations and subcombinations of ranges and specific embodimentstherein are intended to be included.

Preparation of Compound G

In one aspect, provided herein is a method for preparing compound G.Compound G can be prepared in two steps-step (a) and step (b). In step(a), a mixture is formed by admixing together a tertiary amine base anda suspension that includes: (i)(2S,3R)-3-hydroxy-3-(4-methoxyphenyl)-2-((S)-2-(2-morpholino-acetamido)propanamido)propanoicacid (compound “E”):

and

(ii)(S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methyloxiran-2-yl)-1-oxopropan-2-aminiumsalt (compound “F”):

wherein X⁻ is a counterion, in an aprotic solvent to form a mixture. Instep (b), the mixture from step (a) and a coupling agent are admixedtogether at a temperature in a range of about -20° C. to about 25° C. toform compound G.

The counterion (X⁻) can be any anion capable of forming an ionic bondwith the ammonium group of compound F. In some embodiments, X⁻ isselected from the group consisting of tosylate, triflate, acetate,naphthalene sulfonate, 4-nitrobenzenesulfonate, sulfate, methylsulfate,nitrate, fluoride, chloride, bromide, and combinations thereof. In somecases, X⁻ can be tosylate, naphthalene sulfonate, or4-nitrobenzenesulfonate. For example, X⁻ can be tosylate.

The aprotic solvent can be any aprotic solvent (or mixture of solvents)in which the nucleophilic acyl substitution reaction between compounds Eand F can proceed. Suitable aprotic solvents include acetonitrile(“ACN”), dichloromethane (“DCM”), tetrahydrofuran (“THF”),dimethylacetamide (“DMAc”), ethyl acetate (“EtOAc”), isopropyl acetate(“iPrOAc”), dimethylformamide (“DMF”), and combinations thereof. Invarious embodiments, the aprotic solvent is selected from the groupconsisting of ACN, THF, DMF, and DCM. For example, the aprotic solventcan include DCM.

Compound E and compound F can be present in a molar ratio of about 0.8:1 to 1.3:1. In some embodiments, compounds E and F are present in aratio of about 0.9:1 to 1.1:1. For example, the molar ratio of compoundsE and F can be about 1:1, or in a range of 1:1.11 to 1:1.15.

The tertiary amine base can be any tertiary amine base that can promoteor catalyze the nucleophilic acyl substitution reaction betweencompounds E and F. Suitable tertiary amine bases can include, forexample, N,N-diisopropylethylamine (“DIPEA”), triethylamine (“TEA”),N-methylmorpholine (“NMM”), 2,2,6,6-tetramethylpiperidine (“TMP”),2,4,6-trimethylpyridine (“collidine”), and combinations thereof. Forexample, the tertiary amine base can include DIPEA. The tertiary aminebase can be present in a molar ratio to compound E in a range of about1:1 to about 4:1. In some embodiments, the tertiary amine base andcompound E are present in a ratio of about 2.5:1 to 4:1 or 2.5:1 to3.5:1. For example, the ratio of tertiary amine base to compound E canbe about 3.5:1 or 3.9:1.

The coupling agent can include, for example, a carbodiimide reagent, aphosphonium reagent, a uronium reagent, an immonium reagent, animidazolium reagent, an organophosphorus reagent, an acid chloridereagent, a chloroformate reagent, a pyridinium reagent, or combinationsthereof. See, e.g., Han & Kim, Tetrahedron Report 60:2447-2467 (2004);Montalbetti andn Falque, Tetrahedron 61:10827-10852 (2005). Thecarbodiimide can include, for example, N,N’dicyclohexylcarbodimide(“DCC”), 1,3-diisopropylcarbodiimide (“DIC”),1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (“EDC”), or andisopropylcarbodimide (“CIC”), and combinations thereof. The phosphoniumagent can include, for example,(benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate(“BOP”) or benzotriazol-1-yl-oxytripyrrolidinophosphoniumhexafluorophosphate (“PyBOP”), and combinations thereof. The uroniumagent can include, for example,1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid hexafluorophosphate (“HATU”),O-(Benzotriazol-l-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate(“HBTU”), and combinations thereof. For example, the uranium agent caninclude HATU. The imidazolium agent can include, for example,1,1’-carbonyldiimidazole (“CDI”). The acid chloride agent include, forexample, pivaloyl chloride, 2,4, 6-trimethylbenzoyl chloride, andcombinations thereof. The chloroformate agent can include, for example,ethyl chloroformate, isobutyl chloroformate, and combinations thereof.The coupling agent can be present in a molar ratio to compound E in arange of about 0.8:1 to about 1:5. In some embodiments, the couplingagent and compound E are present in a ratio of about 0.9:1 to 1.1:1. Forexample, the ratio of the coupling agent to compound E can be about 1:1or 1.11:1.

The coupling reaction can be performed in the presence of a couplingadditive. Coupling additives are known in the art and any suitable onecan be used for the formation of compound G. Suitable coupling additivesinclude, for example, benzotriazoles, dicarboximides, and succinimides.In some embodiments, the coupling additives is selected from the groupconsisting of N-hydroxysuccinimide (“HOSu”),N-hydroxy-5-norbornene-2,3-dicarboximide (“HONB”),1-hydroxybenzotriazole (“HOBt”), 6-chloro-1-hydroxybenzotriazole(“Cl—HOBt”), 1-hydroxy-7-azabenzotriazole (“HOAt”), and combinationsthereof. For example, the coupling additive can include HOBt.

The temperature of each admixing step is maintained in a range of about-20° C. to about 25° C. In some embodiments, the temperature of eachadmixing step is maintained in a range of about -15° C. to about 25° C.In some cases, the temperature of each admixing step is maintained in arange of about -5° C. to about 15° C. For example, the temperature ofeach admixing step can be maintained in a range of about -5° C. to about5° C. The temperature of each admixing step can be the same ordifferent.

In step (a) of the preparation of compound G, the admixing can occur fora time period of up to about 30 minutes (e.g., up to about 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 16, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, or 30 minutes). In some embodiments, the admixing of step(a) can occur for up to about 10 minutes. In some cases, the admixing ofstep (a) can occur for at least about 30 seconds or at least about 1minute (e.g., at least about 2, 3, 4, 5, 6, 7, 8 or 9 minutes). Forexample, the admixing of step (a) can occur for about 30 seconds toabout 30 minutes, or for about 1 minute to about 20 minutes, or forabout 2 minutes to about 15 minutes, or for about 5 minutes to about 10minutes.

In step (b) of the preparation of compound G, the admixing can occur fora time period of up to about 3 hours (e.g., up to about 1, 1.5, 2, 2.5,or 3 hours). In some embodiments, the admixing of step (b) can occur forup to about 2 hours. In some cases, the admixing of step (b) can occurfor at least about 30 minutes, or at least about 1 hour, or at leastabout 1.5 hours. For example, the admixing of step (b) can occur forabout 30 minutes to about 3 hours, or for about 30 minutes to about 2.5hours, or for about 1 hour to about 2 hours.

In step (b), the coupling reaction can occur under a nitrogenatmosphere. In some cases, the coupling reaction does not occur under anitrogen atmosphere.

After step (b), compound G can be washed with one or more solvents. Thetemperature during washing can optionally be in a range of about 0 °toabout 25° C., or about 15° C. to about 25° C. Suitable solvents forwashing include, for example, water, potassium phosphate monobasic,sodium bicarbonate, sodium sulfate, and combinations thereof. In someembodiments, water is added to compound G after step (b), and theresulting biphasic mixture is separated into an aqueous layer and anorganic layer before washing. In various cases, compound G can be washedwith each of water, potassium phosphate monobasic, sodium bicarbonate,and sodium sulfate.

For example, compound G can be prepared by (a) admixing togethercompound E and compound F (1:1 molar ratio) in DCM for a time period ofup to about 10 minutes to form a mixture, and (b) admixing the mixturefrom step (a) and about 1 molar equivalent of HATU for up to about twohours under a nitrogen atmosphere, wherein the temperature of each stepis in a range of about -20° C. to about 25° C., or about -20° C. to 0°C. The resulting mixture can be quenched with water to result in abiphasic mixture. The organic layer can be separated, washed with water,then with potassium phosphate monobasic, followed by sodium bicarbonateand sodium sulfate (sequentially).

Preparation of Compound E

Compound E can be prepared by admixing a reductant and benzyl(2S,3R)-3-hydroxy-3-(4-methoxyphenyl)-2-((S)-2-(2-morpholinoacetamido)propanamido)propanoate(compound “D”)

The reductant can be any suitable agent capable of removing the benzylgroup on compound D to form the carboxylic acid of compound E. Suitablereductants include, for example, H₂, in the presence of Pd/C orPd(OH)₂/C; Li; Na; lithium 4,4’-di-tert-butylbiphenyl (“Li DTBBP”), andcombinations thereof. For example, the reductant can be H₂, in thepresence of Pd/C.

The admixing of the reductant and compound D can occur in any solventcapable of allowing the reduction reaction to occur. For example, thesolvent can include THF, methanol, or a combination thereof.

In some embodiments, compound D is provided under a nitrogen atmosphereprior to exposure to a hydrogen atmosphere. In various embodiments, thehydrogen atmosphere is established at about 15 psi.

The admixing of the reductant and compound D can occur for a time periodof at least 30 minutes and up to about 5 hours (e.g., up to about 2,2.5, 3, 3.5, 4, or 4.5 hours). In some embodiments, the admixing canoccur for up to about 4 hours. In some cases, the admixing of thereductant and compound D can occur for at least about 30 minutes, or atleast about 1 hour (e.g., at least about 1.5, or 2, or 2.5, or 3, or 3.5hours). For example, the admixing can occur for about 30 minutes toabout 5 hours, or about 1 hour to about 4 hours, or about 2 hour toabout 4 hours.

The temperature of the admixing is maintained in a range of about 10° C.to about 20° C. In some embodiments, the temperature is maintained atabout 17° C.

In some cases, after completion of the admixing, compound E is filtered,such as through diatomaceous earth (i.e., diatomite). The resultingfiltrate can be subsequently washed with a suitable solvent (e.g.,water, methanol, water, and combinations thereof).

Compound E (with or without washing) can be crystallized to form apolymorph, characterized by the differential scanning calorimetry(“DSC”) thermogram, thermogravimetric analysis (“TGA”) data, and X-raypower diffraction (“XRPD”) pattern depicted in FIGS. 2, 3, and 4 ,respectively. For example, the crystallization of compound E can occurin THF and water by heating compound E to a temperature in a range ofabout 50° C. to about 70° C., or about 60° C. to about 70° C., or about55° C. to about 65° C., and then cooling the temperature to about 0° C.Thus, another aspect of the present disclosure is a crystalline form ofcompound E, which is characterized by an XRPD pattern comprising peaksat 6.2, 8.5, 9.7, 12.7, 13.7, 16.0, 16.9 17.2, 18.4, 18.9, 19.2, 19.7,22.5, 24.7, 25.4, 28.7, and 29.7 ± 0.2° 2θ using Cu Kα radiation, asshown in FIG. 4 .

For example, compound E can be prepared by admixing together areductant, such as H₂, in the presence of Pd/C, and compound D under anitrogen atmosphere, at 10° C. to 20° C., for a time period of at least30 minutes up to 4 hours. Compound E can be filtered across diatomite,and the resulting filter cake can be washed (e.g., with water, methanol,and/or THF). Compound E can be crystallized by heating it to about 60°C. to 70° C., adjusting the temperature to about 55° C. to 65° C. andadding THF to mixture, heating the mixture back to 60° C. to 70° C.,adding water to the heated mixture, cooling the mixture back to 55° C.to 65° C., adding a seed crystal to the mixture, and stirring the seededmixture for about two hours at 0° C. Filtering, washing, and drying thecooled mixture results in crystalized compound E.

Preparation of Compound F

In another aspect, provided herein is a method of preparing compound F.

wherein X⁻ is a counterion.

Compound F can be prepared in three steps-steps (a), (b), and (c). Instep (a), a mixture is formed by admixing together an aprotic solvent,trifluoroacetic acid (“TFA”), andtert-butyl-((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methyloxiran-2-yl)-1-oxopropan-2-yl)carbamate(compound H):

at a temperature in a range of about -5° C. to about 5° C. In step (b),the mixture from step (a) is concentrated. In step (c), the concentratedmixture of step (b) is admixed together with an acid at a temperature ina range of about -5° C. to 5° C. to form compound F.

The acid can be any acid capable of forming a salt with the amniniumgroup of compound F. Suitable acids include, for example,p-toluenesulfonic acid, trifluoromethanesulfonic acid, acetic acid,trifluoroacetic acid, naphthalene sulfonic acid, 4-nitrobenzenesulfonicacid, sulfonic acid, methylsulfonic acid, benzenesulfonic acid, nitricacid, HF, HCl, HBr, and combinations thereof. In some embodiments, theacid is selected from the group consisting ofp-toluenesulfonic acid,naphthalene sulfonic acid, 4-nitrobenzenesulfonic acid, and combinationsthereof. For example, the acid can include p-toluenesulfonic acid.

The aprotic solvent in step (a) can be any aprotic solvent (or mixtureof solvents) in which the reaction can proceed. Suitable aproticsolvents can include acetonitrile (“ACN”), dichloromethane (“DCM”),tetrahydrofuran (“THF”), dimethylacetamide (“DMAc”), methyl tert-butylether (“MTBE”), isopropyl ether (“IPE”), and combinations thereof. Forexample, the aprotic solvent can include DCM.

The trifluoroacetic acid in step (a) can be present in a molar ratio tocompound H in a range of about 15:1 to 5:1. In some embodiments, thetrifluoroacetic acid and Compound H are present in a ratio of about 10:1to 7.5:1. For example, the molar ratio of trifluoroacetic acid andcompound H can be about 8:1.

In some embodiments, the deprotection reaction of step (a) occurs undera nitrogen atmosphere.

The temperature of the mixture in step (a), step (c), or both step (a)and step (c) is maintained in a range of about -5° C. to about 5° C., orat about 0° C. In some embodiments, the mixture is concentrated in step(b) at a temperature in a range of about 15° C. to about 25° C.

In some cases, the admixing of step (a) can occur for a time period ofat least 30 minutes up to about 3 hours (e.g., up to about 1, 1.5, 2,2.5, or 3 hours). In some embodiments, the admixing of step (a) canoccur for a time period of up to about 2 hours. In some cases, theadmixing of step (a) can occur for at least about 30 minutes, or atleast about 1 hour, or at least about 1.5 hours. For example, theadmixing of step (a) can occur for about 30 minutes to about 3 hours, orabout 30 minutes to about 2.5 hours, or about 1 hour to about 2 hours.

In various cases, the admixing of step (c) can occur for a time periodof at least 5 hours up to about 12 hours (e.g., up to about 7, 8, 9, 10,or 11 hours). In some embodiments, the admixing of step (c) can occurfor a time period of up to about 10 to 12 hours. In some cases, theadmixing of step (c) can occur for at least about 5 hours (e.g., atleast about 6, 7, 8, 9, or 10 hours). For example, the admixing of step(c) can occur for about 5 hours to about 12 hours, or about 10 hours toabout 12 hours.

In some cases, the concentrated mixture of step (b) can be rinsed with apolar, aprotic solvent. Suitable polar, aprotic solvents include, forexample, diethyl ether, tetrahydrofuran (“THF”), acetonitrile (“ACN”),methyl tert-butyl ether (“MBTE”), isopropyl ether (“IPE”)andcombinations thereof. For example, the polar, aprotic solvent can beMBTE.

After step (c), compound F can optionally be filtered at a temperaturein a range of about -5° C. to about 5° C., washed with one or morepolar, aprotic, solvents (e.g., diethyl ether, tetrahydrofuran (“THF”),acetonitrile (“ACN”), methyl tert-butyl ether (“MBTE”), isopropyl ether(“IPE”), and combinations thereof), and/or dried.

Compound F can be crystallized to form a polymorph, characterized by thedifferential scanning calorimetry (“DSC”) thermogram, thermogravimetricanalysis (“TGA”) data, and X-ray power diffraction (“XRPD”) patterndepicted in FIGS. 5, 6, 7, 8, and 9 . Thus, another aspect of thepresent disclosure is a crystalline form of compound F, such as thetosylate salt of compound F, which is characterized by an XRPD patterncomprising peaks at 6.8, 7.1, 7.4, 14.2, 14.8, 17.0, 17.5, 17.8, 18.5,18.7, 20.1, 20.3, 23.0, 23.6, 24.5, 29.3, and 31.2 ± 0.2° 2θ using Cu Kαradiation, as shown in FIG. 9 .

The tosylate form of compound F also can be characterized by a singlecrystal X-ray diffraction (“XRD”) structure, as described in theExamples section below. The crystal, as represented in FIG. 10 , has aunit cell dimension of a = 13.264(3) Å, a= 90°, b = 5.6920(11) Å, b=109.410(4)°, c = 13.416(3) Å, g = 90° and belongs to the space group P21. The Flack parameter is 0.03 (0.08 su). Crystallizations that usedother acids such as 2-napthalenesulfonic, methanesulfonic,benzenesulfonic, phosphoric, and sulfuric acid did not provide x-rayquality crystals in the following solvents: toluene, diethyl ether,MTBE, 1,4-dioxane, ethyl acetate, acetone, acetonitrile, butanol,isopropanol, and hexane/ethyl actate (1:1 ratio).

For example, compound F can be prepared by (a) admixing together anaprotic solvent (e.g., DCM), TFA, and compound H at a molar ratio of 8:1and a temperature of about 0° C., under a nitrogen atmosphere, for atime period of up to 2 hours, (b) concentrating the mixture at atemperature of about 15° C. to 25° C., and (c) admixing the concentratedmixture and an acid (e.g., p-toluenesulfonic acid) at temperature ofabout 0° C. for a time period of 10 to 12 hours. The resulting compoundF can be filtered at about 0° C., washed with a polar, aprotic solvent(e.g., MBTE), and dried under vacuum.

Preparation of Compound D

In another aspect, provided herein is method for preparing compound D.

Compound D can be prepared in two steps-step (a) and step (b). In step(a), a mixture is prepared by admixing together a tertiary amine baseand a suspension of compound B and compound C in an aprotic solvent: (i)(2S,3R)-1-(benzyloxy)-3-hydroxy-3-(4-methoxyphenyl)-1-oxopropan-2-aminiumsalt (compound “B”):

wherein X⁻ is a counterion, and

(ii) morpholinoacetyl)-L-alanine (compound “C”):

In step (b), the mixture from step (a) and a coupling agent are admixedtogether at a temperature in a range of about -5° C. to about 5° C. toform compound D.

The counterion (X⁻) can be any anion capable of forming an ionic bondwith the ammonium group of compound B. In some embodiments, X⁻ isselected from the group consisting of tosylate, triflate, acetate,naphthalene sulfonate, 4-nitrobenzenesulfonate, sulfate, methylsulfate,nitrate, fluoride, chloride, bromide, and combinations thereof. In somecases, X⁻ can be tosylate, naphthalene sulfonate, or4-nitrobenzenesulfonate. For example, X⁻ can be chloride.

The aprotic solvent can be any aprotic solvent (or mixture of solvents)in which the nucleophilic acyl substitution reaction between compounds Band C can proceed. Suitable aprotic solvents can include acetonitrile(“ACN”), dichloromethane (“DCM”), tetrahydrofuran (“THF”),dimethylacetamide (“DMAc”), ethyl acetate (“EtOAc”), isopropyl acetate(“iPrOAc”), dimethylformamide (“DMF”), and combinations thereof. Forexample, the aprotic solvent can include ACN.

Compound B and compound C can be present in a molar ratio of about0.65:1 to 1.1:1. In some embodiments, compounds B and C are present in aratio of about 0.75: 1 to 1:1. For example, the molar ratio of compoundsB and C can be about 0.8:1.

The tertiary amine base can be any tertiary amine base that can promoteor catalyze the nucleophilic acyl substitution reaction betweencompounds B and C. Suitable tertiary amine bases can include, forexample, N,N-diisopropylethylamine (“DIPEA”), triethylamine (“TEA”),N-methylmorpholine (“NMM”), 2,2,6,6-tetramethylpiperidine (“TMP”),2,4,6-trimethylpyridine (“collidine”), or combinations thereof. Forexample, the tertiary amine base can include DIPEA. The tertiary aminebase can be present in a molar ratio to compound B in a range of about1:1 to about 3.5:1. For example, the molar ratio of tertiary amine baseto compound B can be about 3.5:1.

The coupling agent can include, for example, a carbodiimide reagent, aphosphonium reagent, a uronium reagent, an immonium reagent, animidazolium reagent, an organophosphorus reagent, an acid chloridereagent, a chloroformate reagent, a pyridinium reagent, or combinationsthereof, as previously described above for the preparation of compoundG. Examples of the carbodiimide reagent, phosphonium reagent, uroniumreagent, immonium reagent, imidazolium reagent, organophosphorusreagent, acid chloride reagent, chloroformate reagent, and pyridiniumreagent are described above for the preparation of compound G. In someembodiments, the uronium agent can include HATU, HBTU, and combinationsthereof. For example, the uranium agent can be HATU. The coupling agentcan be present in a molar ratio to compound B in a range of about 1:1 toabout 1:3. In some embodiments, the coupling agent and compound B arepresent in a ratio of about1: 1 to 1:2. For example, the ratio of thecoupling agent to compound B can be about 1:1.5.

The coupling reaction can be performed in the presence of a couplingadditive. Examples of coupling additives amounts thereof are describedfor the preparation of compound G.

The temperature of each admixing step is maintained in a range of about-5° C. to about 5° C. In some embodiments, the temperature of eachadmixing step is maintained at about 0° C. The temperature of eachadmixing step can be the same or different.

In step (b) of the preparation of compound D, the admixing can includemixing portions of the coupling agent with the mixture from step (a)over a time period of at least 1 minute up to about 30 minutes (e.g., upto about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 16, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, or 30 minutes). In some embodiments,portions of the coupling agent can be added to the mixture from step (a)over a time period of at least about 1 minute (e.g., at least about 2,3, 4, 5, 6, 7, 8 , 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25 minutes). For example, portions of the coupling agent canbe added to the mixture from step (a) for a time period of about 1minute to about 30 minutes or about 10 minutes to about 30 minutes, orabout 20 minutes to about 30 minutes. The admixing of step (b) can alsoinclude stirring the mixture for up to about 3 hours (e.g., up to about1, 1.5, 2, 2.5, or 3 hours). In some embodiments, the stirring can occurfor up to about 2 hours. In some cases, the stirring can occur for atleast about 30 minutes, or at least about 1 hour, or at least about 1.5hours. For example, the stirring can occur for about 30 minutes to about3 hours, or about 30 minutes to about 2.5 hours, or about 1 hour toabout 2 hours.

After step (b), compound D can be quenched and/or washed with one ormore solvents at a temperature in a range of about 15° C. to about 25°C. Suitable solvents for the quenching and/or washing include, forexample, water, isopropyl acetate, potassium phosphate monobasic, sodiumbicarbonate, sodium sulfate, THF, and combinations thereof.

For example, compound D can be prepared by (a) admixing togethercompound B and compound (C) (1:1 molar ratio) and a tertiary amine base(e.g., DIPEA) in ACN, and (b) admixing the mixture from step (a) andabout 1 molar equivalent of HATU in portions over a time period of about30 minutes, and then stirring the mixture for a time period of up toabout 2 hours, wherein the temperature of each step is about 0° C. Theresulting mixture from step (b) can be quenched with, for example,sodium bicarbonate to form a biphasic mixture. The organic phase can beseparated and washed with sodium bicarbonate, potassium phosphatemonobasic, and/or sodium sulfate.

Preparation of Compound B

Compound B can be prepared by admixing (i) an acid and (ii) benzyl(2S,3R)-2-((tert-butoxycarbonyl)amino)-3-hydroxy-3-(4-methoxyphenyl)propanoate(compound “A”):

in an aprotic solvent.

The acid can be any suitable acid capable of deprotecting the aminogroup on compound A. Suitable acids include, for example,p-toluenesulfonic acid, trifluoromethanesulfonic acid, acetic acid,trifluoroacetic acid, naphthalene sulfonic acid, 4-nitrobenzenesulfonicacid, sulfonic acid, methylsulfonic acid, nitric acid, HF, HCl, HBr, andcombinations thereof. In some embodiments, the acid includestrifluoroacetic acid or HCl.

The aprotic solvent can be any solvent in which the deprotectionreaction can occur. Suitable solvents include ethyl acetate,N-methylpyrrolidone (“NMP”), tetrahydrofuran (“THF”), acetone,dimethylformamide (“DMF”), acetonitrile (“ACN”), dimethyl sulfoxide(“DMSO”), dicholormethane (“DCM”), and combinations thereof. Forexample, the solvent can include ethyl acetate, DCM, or a combinationthereof.

In some embodiments, the temperature of the mixture during the admixingstep is maintained in a range of about 15° C. to about 25° C., or atabout 20° C.

In some cases, after completion of the admixing, compound B is filteredand dried under vacuum to form a crystalline polymorph, which ischaracterized by the DSC thermogram depicted in FIG. 1 . Thus, anotheraspect of the present disclosure is a crystalline form of compound B,which is characterized by an XRPD pattern comprising peaks at 4.6, 9.2,13.8, 18.5, and 32.9 ± 0.2° 2θ using Cu Kα radiation.

For example, compound B can be prepared by admixing together an acid(e.g., HCl) and compound A at 20° C., filtering, and drying theresulting compound B.

EXAMPLES

The following examples are provided for illustration and are notintended to limit the scope of the invention.

General Synthetic Scheme

Compound G can be prepared according to Scheme 1, shown above.

Example 1: Large-Scale Preparation of the HCl salt of(2S,3R)-1-(benzyloxy)-3-hydroxy-3-(4-methoxyphenyl)-1-oxopropan-2-aminiumsalt (Compound “B”):

Ethyl acetate (58.5 kg) at 20° C. was charged with HCl gas (6.8 kg). Tothis solution was dissolved benzyl(2S,3R)-2-((tert-butoxycarbonyl)amino)-3-hydroxy-3-(4-methoxyphenyl)propanoate(compound “A”):

(5 kg, 12.5 mol, pre-dissolved in 32.5 kg ethyl acetate). The suspensionwas stirred at 20° C. and upon completion, as determined by HPLC, wasfiltered and dried under vacuum at 45° C. to provide a crystallinepolymorph of compound B (3.85 kg) as the HCl salt. LC/MS (LRMS(MH) m/z:302). HPLC Purity 97.9%. The characteristic DSC curve is shown in FIG. 1.

Example 2: Small-Scale Synthesis of the TFA Salt of(2S,3R)-1-(benzyloxy)-3-hydroxy-3-(4-methoxyphenyl)-1-oxopropan-2-aminiumSalt (Compound “B”)_(:)

Trifluoroacetic acid (“TFA”) (20 mL) was added to a solution of compoundA (7.0 g, 17.4 mmol) in dicholormethane (“DCM”) (50 mL) at 0° C. Themixture was stirred for 30 min then diluted with DCM (100 mL). SaturatedNaHCO₃ (aqueous, 100 mL) was added and the two layers were separated.The aqueous layer was extracted with DCM (2 x 100 mL) and the combinedorganic layers were dried over anhydrous sodium sulfate thenconcentrated to afford crude compound B (5.0 g, 84% yield) as the TFAsalt. LC/MS (LRMS(MH) m/z: 302.

Example 3: Large-Scale Preparation of(2S3R)-3-hydroxy-3-(4-methoxyphenyl)-2-((S)-2-(2-morpholinoacetamido)propanamido)propanoate(Compound “D”)

To compound B (3.8 kg) and (2-morpholinoacetyl)-L-alanine (compound“C”):

(2.5 kg) at 20° C. was added acetonitrile (30.4 kg). The temperature wasadjusted to 0° C. and N,N-diisopropylethylamine (“DIPEA”) (3.19 kg) wasadded, followed by1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid hexafluorophosphate (“HATU”) (5.22 kg) portion-wise over 30 min.The reaction mixture was stirred for 2 h at 0° C. then quenched with3.5% NaHCO₃ (aqueous, 46 kg) and stirred for 30 min. After standing for1 h at 20° C., NaHCOs solid was added and the mixture was stirred for 30min then allowed to stand again at 20° C. for 1 h. The aqueous layer wasdiluted with water (30.6 kg), extracted with isopropyl acetate(“iPrOAc”) (23.4 kg), and the organic layers were combined. The organiclayers were chased with iPrOAc (3 x 27 kg), washed with 3.5% NaHCO₃(aqueous, 30 kg), KH₂PO₄ (aqueous, 3 x 65 kg), water (15 kg), 7% NaHCO₃(aqueous, 2 x 61 kg), and 5% Na₂SO₄ (aqueous, 3 x 55 kg). The solutionwas concentrated to 18 L then chased with tetrahydrofuran (“THF”) (4 x22.8 L) to provide the product (5.04 kg, 90% yield, 97.9% purity byHPLC) as a solution in THF (34.5 wt%, 14.6 kg total).

Similar results were obtained using1-ethyl-3-(3-dimethylaminopropyl)carbodiimide HCl (“EDC”) (1.1 equiv.)in place of HATU as a coupling reagent.

Example 4: Small-Scale Synthesis of Compound D

The reagents HATU (6.79 g, 17.9 mmol) and DIPEA (9.63 mL, 59.2 mmol)were added to a solution of compound B (TFA salt, 5.0 g, 14.8 mmol) andcompound C (3.36 g, 15.9 mmol) in dimethylformamide (“DMF”) (100 mL) at0° C. The reaction mixture was allowed to warm to room temperature andstirred for 1 h. The mixture was concentrated and the residue waspurified by flash column chromatography on silica gel (petroleumether/EtOAc = 2:1 to 1:2) to afford compound D (5.8 g, 78% yield) as acolorless solid. LC/MS (LRMS(MH) m/z: 500.

Example 5: Large-Scale Preparation of(2S3R)-3-hydroxy-3-(4-methoxyphenyl)-2-((S)-2-(2-morpholino-acetamido)propanamido)propanoicacid (compound “E”)_(:)

To a solution of benzyl(2S,3R)-3-hydroxy-3-(4-methoxyphenyl)-2-((S)-2-(2-morpholinoacetamido)propanamido)propanoate(compound “D”) (5.04 kg as a 34.5% wt solution in THF) was added THF(3.25 kg) followed by methanol (7.0 kg). A N₂ atmosphere was establishedwithin the reaction vessel and Pd/C (10%, 473 g) was added undernitrogen protection. THF (500 g) and methanol (1 kg) were added to washthe reaction vessel and an H₂ atmosphere was established (15 psi). Thereaction was stirred for 4 h at 17° C. then filtered across diatomite.The wet cake was washed with methanol (30 kg), concentrated to 3-4volume, chased with THF (4 x 45 kg), and heated to 60-70° C. After 2 hthe temperature was adjusted to 50-60° C. and THF (30 kg) was added. Themixture was again heated to 60-70° C. for 2 h. To this solution wasadded water (370 kg) at 60-70° C., then the mixture was cooled to 55-65°C. Seed crystal (18.0 g) was added and the mixture was stirred at 55-65°C. for 1 h. Twice the suspension was concentrated to 5-6 volumes andstirred for 2 h at 0° C. The mixture was filtered using THF (10 kg) towash. The wet cake was dried to provide a crystalline polymorph ofcompound E (3.54 kg, 97.6 % purity). Characteristic DSC, TGA, and XRPDdata is shown in FIGS. 2-4 .

Example 6: Small-Scale Synthesis of Compound E

To a solution of compound D (5.8 g, 11.6 mol) in THF (120 mL) was addedPd/C (1.5 g, 10%). The mixture was stirred under an H₂ atmosphere (1atm) at ambient temperature overnight then filtered through a pad ofcelite. The filtrate was concentrated under reduced pressure and theresidue was washed with EtOAc (20 mL) to afford Compound E (4.8 g, ~100%yield) as a colorless solid.

Example 7: Large-Scale Preparation of the Tosylate Salt of(S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methyloxiran-2-yl)-1-oxopropan-2-aminiumsalt (compound “F”)

To a solution of tert-butyl((S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methyloxiran-2-yl)-1-oxopropan-2-yl)carbamate(compound “H”):

(134 g) in DCM (402 mL) at 0° C. was added TFA (414.3 g, 8 eq) at a rateto maintain the internal temperature at -5-5° C. The reaction mixturewas stirred for 2 h at this temperature under N₂. The dark mixture wasthen concentrated to remove DCM and TFA at 15-25° C. The solution waschased with methyl tert-butyl ether (“MBTE”) (5 x 2 L). HPLC analysisindicated 2.72 eq TFA remained in the solution and MTBE (804 mL) wasadded at 15-25° C. To this solution at 0° C. was added p-toluenesulfonicacid (“PTSA”) (83.6 g) and the mixture was stirred at this temperaturefor 10-12 h. The mixture was then filtered at 0° C., washed with MTBE (3x 268 mL then 1 x 168 mL), and the filter cake was dried under vacuum at15-25° C. for 16-18 h to provide compound F (126 g, 99.4% purity) as thetosylate salt. Characteristic DSC and TGA curves are shown in FIGS. 5and 6 , and a characteristic XRPD pattern can be found in FIG. 9 .

Example 8: Small-Scale Synthesis of the Naphthalene Sulfonate Salte ofCompound F

To compound H (2 g) was added DCM (8 mL) and the mixture was cooled to5° C. TFA (8 mL) was added at a rate to maintain the internaltemperature below 10° C. The mixture was then stirred at ambienttemperature for 30 min then concentrated under vacuum. Toluene (3 x 5mL) was added to remove excess TFA. To the TFA salt was added EtOAc (4mL) followed 2-napthalenesulfonic acid (6.78 mmol, 1.41 g, dissolved in10 mL EtOAc). The mixture was stirred at ambient temperature and acolorless solid precipitated within 5 min. The mixture was stirred anadditional 15 min then filtered using EtOAc (10 mL) to rinse. The solidwas placed under vacuum for 16 h to provide the naphthalene sulfonatesalt as a colorless solid (1.62 g, 72% yield). Characteristic DSC andTGA data are shown in FIGS. 7 and 8 .

A similar procedure was used to generate the 4-nitrobenzenesulfonic acidof Compound F.

Example 9: Large-Scale Preparation of(2S,3R)-N-[(2S)-3-(cyclopent-1-en-1-yl)-1-[(2R)-2-methyloxiran-2-yl]-1-oxopropan-2-yl]-3-hydroxy-3-(4-methoxyphenyl)-2-[(2S)-2-[2-(morpholin-4-yl)acetamido]propanamido]propanamide(Compound “G”)

To Compound E (110.0 g) and Compound F (tosylate salt, 110.0 g) wasadded DCM (1.46 kg) and the suspension was cooled to -15 to -5° C. DIPEA(122.1 g) was added at a rate to maintain an internal temperature of -15to -5° C. The mixture was then stirred for 10 min and to this solutionwas added HATU (114.4 g) at -15 to -5° C. under nitrogen. The mixturewas stirred for 2 h at -15 to -5° C. and compound F (3.91 g) was added.After 10 min, the internal temperature was adjusted to 5 to 15° C. thenwater (1100 g) was added. The solution was stirred for 30-60 min andallowed to stand for 30-60 min. The organic layer was separated andwashed with water (1100 g) for 30-60 min and the mixture was allowed tostand for 30-60 min. The organic phases were combined and thetemperature was raised to 15 to 25° C. The solution was concentrated to2-4 volumes under vacuum (< 45° C.). iPrOAc (957 g) was added and thesolution was concentrated to 2-4 volumes under vacuum (< 45° C.). Thesolution was washed with 5% KH₂PO4 (aqueous, 1100 g), 1% KH₂PO4(aqueous, 2 x 1100 g), 7% NaHCO₃ (aqueous, 1100 g), and 5% Na₂SO₄(aqueous, 1100 g). The product was provided as a 10.53 wt% solution iniPrOAc (89.8%).

Example 10: Small-Scale Preparation of Compound G

HATU (5.35 g, 14.1 mmol) and DIPEA (9.55 mL, 58.7 mmol) were added to asolution of compound E (4.8 g, 11.7 mmol) and compound F (TFA salt, 3.46g, 11.7 mmol) in DMF (90 mL) at 0° C. The reaction mixture was allowedto warm to room temperature and stirred for 30 min. The mixture wasconcentrated and the residue was purified by flash column chromatographyon silica gel (petroleum ether/EtOAc = 2:1 to EtOAc) to afford CompoundG (4.8 g, 70% yield, 95.2% purity by HPLC) as a colorless solid. LC/MS(LRMS(MH) m/z: 587).

Example 11: Preparation of a Single Crystal of the Tosylate Salt ofCompound F

The TFA salt of compound F (0.70 g, 2.39 mmol) was dissolved in MTBE(3.5 mL) and p-toluenesulfonic acid (0.45 g, 2.39 mmol) was added. Thesolution was sealed in a vial and allowed to stand at ambienttemperature. After 9 months, the solvent was removed from theprecipitated crystals and the solid was allowed to dry at ambienttemperature over 2 days. See Flack, H. D.; Bernardinelli, G. The Use ofX-Ray Crystallography to Determine Absolute Configuration. Chirality,2008, 20, 681-690.

Example 12: Comparison of Stability of Trifluoroacetic Acid and TosylateSalts of Compound F

The stability of the trifluoroacetic and tosylate salts of compound Fwere determined by exposing multiple lots of each salt to a temperatureof 25° C. at a relative humidity of 40% for one or ninety days, anddetermining the percentage that each sample had decomposed. As shown inthe table below, the tosylate salt of compound F is significantly morestable than its trifluoroacetic acid counterpart.

Form Lot # % Purity at t = 0 % Purity at Indicated Time (days) %Decomposition Tosylate 1 97.2 96.0 (90) 1.2 2 88.6 85.0 (90) 3.6Trifluoroacetate 3 85.3 79.6 (1) 5.7 4 94.8 86.0 (1) 8.8

The foregoing description is given for clearness of understanding only,and no unnecessary limitations should be understood therefrom, asmodifications within the scope of the invention may be apparent to thosehaving ordinary skill in the art.

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise” and variations such as“comprises” and “comprising” will be understood to imply the inclusionof a stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integers or steps.

Throughout the specification, where compositions are described asincluding components or materials, it is contemplated that thecompositions can also consist essentially of, or consist of, anycombination of the recited components or materials, unless describedotherwise. Likewise, where methods are described as including particularsteps, it is contemplated that the methods can also consist essentiallyof, or consist of, any combination of the recited steps, unlessdescribed otherwise. The invention illustratively disclosed hereinsuitably may be practiced in the absence of any element or step which isnot specifically disclosed herein.

The practice of a method disclosed herein, and individual steps thereof,can be performed manually and/or with the aid of or automation providedby electronic equipment. Although processes have been described withreference to particular embodiments, a person of ordinary skill in theart will readily appreciate that other ways of performing the actsassociated with the methods may be used. For example, the order ofvarious of the steps may be changed without departing from the scope orspirit of the method, unless described otherwise. In addition, some ofthe individual steps can be combined, omitted, or further subdividedinto additional steps.

All patents, publications and references cited herein are hereby fullyincorporated by reference. In case of conflict between the presentdisclosure and incorporated patents, publications and references, thepresent disclosure should control.

1-42. (canceled)
 43. A method of preparing benzyl(2S,3R)-3-hydroxy-3-(4-methoxyphenyl)-2-((S)-2-(2-morpholinoacetamido)propanamido)propanoate(compound “D”)

comprising: (a) admixing a tertiary amine base and a suspension of: (i)(2S,3R)-1-(benzyloxy)-3-hydroxy-3-(4-methoxyphenyl)-1-oxopropan-2-aminiumsalt (compound “B″):

wherein X⁻ is a counterion; and (ii) (2-morpholinoacetyl)-L-alanine(compound “C”):

in an aprotic solvent to form a mixture; and (b) admixing a couplingagent and the mixture of step (a) to form compound D; wherein thetemperature of each admixing step is maintained at -5° C. to 5° C. 44.The method of claim 43, wherein X⁻ is selected from the group consistingof tosylate, triflate, acetate, naphthalene sulfonate,4-nitrobenzenesulfonate, sulfate, methylsulfate, nitrate, fluoride,chloride, bromide, and combinations thereof.
 45. (canceled)
 46. Themethod of claim 43, wherein the aprotic solvent is selected from thegroup consisting of acetonitrile (“ACN”), dichloromethane (“DCM”),tetrahydrofuran (“THF”), dimethylacetamide (“DMAc”), and combinationsthereof.
 47. (canceled)
 48. The method of claim 43, wherein the tertiaryamine base is selected from the group consisting ofN,N-diisopropylethylamine (“DIPEA”), triethylamine (“TEA”),N-methylmorpholine (“NMM”), 2,2,6,6-tetramethylpiperidine (“TMP”),2,4,6-trimethylpyridine (“collidine”), and combinations thereof.
 49. Themethod of claim 48, wherein the tertiary amine base comprises DIPEA. 50.The method of claim 43, wherein the coupling agent comprises acarbodiimide reagent, a phosphonium reagent, a uronium reagent, animmonium reagent, an imidazolium reagent, an organophosphorus reagent,an acid chloride reagent, a chloroformate reagent, or a pyridiniumreagent.
 51. The method of claim 50, wherein the uronium reagent isselected from the group1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid hexafluorophosphate (“HATU”),O-(Benzotriazol-1-yl)-N,N,N;N′-tetramethyluronium hexafluorophosphate(“HBTU”), and combinations thereof.
 52. (canceled)
 53. The method ofclaim 43, wherein the molar ratio of coupling agent to compound B is 1to
 1. 54. The method of claim 43, wherein the coupling reagent furthercomprises a coupling additive.
 55. The method of claim 54, wherein thecoupling additive is selected from the group consisting of abenzotriazole, a dicarboximide, a succinimide, and combinations thereof.56. The method of claim 55, wherein the coupling additive is selectedfrom the group consisting of N-hydroxysuccinimide (“HOSu”),N-hydroxy-5-norbornene-2,3-dicarboximide (“HONB”),1-hydroxybenzotriazole (“HOBt”), 6-chloro-1-hydroxybenzotriazole(“CI—HOBt”), 1-hydroxy-7-azabenzotriazole (“HOAt”), and combinationsthereof.
 57. The method of claim 43, wherein the temperature of eachadmixing step is maintained at -5° C. to 5° C.
 58. The method of claim43, wherein the admixing of step (b) comprises mixing portions of thecoupling agent with the mixture from step (a) over 30 minutes. 59.(canceled)
 60. The method of claim 43, further comprising washingcompound D with one or more of the following: water, isopropyl acetate,potassium phosphate monobasic, sodium bicarbonate, sodium sulfate, andTHF.
 61. The method of claim 43, wherein compound B is prepared byadmixing (i) an acid and (ii) benzyl(2S,3R)-2-((tert-butoxycarbonyl)amino)-3-hydroxy-3-(4-methoxyphenyl)propanoate(compound “A”):

in a polar, aprotic solvent, to form compound B.
 62. The method of claim61, wherein the acid is selected from the group consisting ofp-toluenesulfonic acid, trifluoromethanesulfonic acid, acetic acid,trifluoroacetic acid, naphthalene sulfonic acid, 4-nitrobenzenesulfonicacid, sulfonic acid, methylsulfonic acid, nitric acid, HF, HCI, HBr, andcombinations thereof.
 63. (canceled)
 64. The method of claim 61, whereinthe polar, aprotic solvent is selected from the group consisting ofethyl acetate, N-methylpyrrolidone (“NMP”), tetrahydrofuran (“THF”),acetone, dimethylformamide (“DMF”), acetonitrile (“ACN”), dimethylsulfoxide (“DMSO”), dicholormethane (“DCM”), and combinations thereof.65. The method of claim 64, wherein the polar, aprotic solvent comprisesethyl acetate, DCM, or combinations thereof.
 66. The method of claim 61,wherein the admixing step comprises stirring at a temperature in a rangeof 15° C. to 25° C.
 67. (canceled)
 68. A crystalline compound selectedfrom: (a) a crystalline form of(2S,3R)-1-(benzyloxy)-3-hydroxy-3-(4-methoxyphenyl)-1-oxopropan-2-aminiumchloride salt (compound “B—Cl”)

characterized by an X-ray powder diffraction pattern comprising peaks at4.6, 9.2, 13.8, 18.5, and 32.9 ± 0.2° 2θ using Cu Kα radiation, (b) acrystalline form of(2S,3R)-3-hydroxy-3-(4-methoxyphenyl)-2-((S)-2-(2-morpholinoacetamido)propanamido)propanoicacid (compound “E”):

characterized by an X-ray powder diffraction pattern comprising peaks at6.2, 8.5, 9.7, 12.7, 13.7, 16.0, 16.9 17.2, 18.4, 18.9, 19.2, 19.7,22.5, 24.7, 25.4, 28.7, and 29.7 ± 0.2° 2θ using Cu Kα radiation; and(c) a crystalline form of(S)-3-(cyclopent-1-en-1-yl)-1-((R)-2-methyloxiran-2-yl)-1-oxopropan-2-aminiumsalt (compound “F”):

wherein X⁻ is tosylate, characterized by an X-ray powder diffractionpattern comprising peaks at 6.8, 7.1, 7.4, 20.3, 23.0, 23.6, 24.5, 29.3,and 31.2 ± 0.2° 2θ using Cu Kα radiation. 69-70. (canceled)